install.packages("Signac") #seurat addon for analyzing chromatin
library(hdf5r) #need to read h5 files
library(Seurat)
library(Signac)
library(ggplot2)
library(patchwork)
setwd("C:\\singlecell_atac")
getwd()
[1] "C:/singlecell_atac"

Pre-processing workflow

counts <- Read10X_h5("GSE190424_RAW\\Aged\\GSM5723632_Aged_HSC_filtered_peak_bc_matrix.h5")
# counts is a feature by cell matrix, here features are coordinates of peaks.
counts # 167771 features x 3694 cells
meta <- read.csv("GSE190424_RAW\\Aged\\GSM5723632_Aged_HSC_singlecell.csv.gz",
                 header=TRUE,
                 row.names = 1) 

meta
  chrom_assay <- CreateChromatinAssay(
  counts = counts,
  sep = c(":", "-"), # features (chr1:3670444-3672753, look in counts) are sep by : and ,
  genome = 'mm10', 
  fragments = 'GSE190424_RAW\\Aged\\GSM5723632_Aged_HSC_fragments.tsv.gz',
  min.cells = 10, # Keep peaks found in ≥10 cells.
  min.features = 200 # Keep cells with ≥200 peaks
)
Checking for 3694 cell barcodes
# Notes:
# 
# Make sure fragments and its index are in same folder
# Names of both files should match except for the .tbi
# for example: see below
list.files("GSE190424_RAW\\Aged")
[1] "GSM5723632_Aged_HSC_filtered_peak_bc_matrix.h5"
[2] "GSM5723632_Aged_HSC_fragments.tsv.gz"          
[3] "GSM5723632_Aged_HSC_fragments.tsv.gz.tbi"      
[4] "GSM5723632_Aged_HSC_singlecell.csv.gz"         

data <- CreateSeuratObject(
  counts = chrom_assay,
  assay = "peaks",
  meta.data = meta
)
data
An object of class Seurat 
148897 features across 3694 samples within 1 assay 
Active assay: peaks (148897 features, 0 variable features)
 2 layers present: counts, data
data[[]]

if (!require("BiocManager", quietly = TRUE))
    install.packages("BiocManager")

BiocManager::install("EnsDb.Mmusculus.v79") # EnsDb.Hsapiens.v86 for human
BiocManager::install("GenomeInfoDb") #translation between chromosome names
BiocManager::install("biovizBase")
library(EnsDb.Mmusculus.v79)
library(GenomeInfoDb)
# get all annotated mouse genes from Ensembl-based annotation database

annotations <- GetGRangesFromEnsDb(ensdb = EnsDb.Mmusculus.v79)
seqlevelsStyle(annotations) <- 'UCSC'

# Note: features have chr1 prefix which is UCSC-style chromosome names. 
annotations
GRanges object with 1763965 ranges and 5 metadata columns:
                     seqnames          ranges strand |              tx_id   gene_name
                        <Rle>       <IRanges>  <Rle> |        <character> <character>
  ENSMUSE00001236884     chr3 3508030-3508332      + | ENSMUST00000108393       Hnf4g
  ENSMUSE00000676606     chr3 3634150-3634347      + | ENSMUST00000108394       Hnf4g
  ENSMUSE00001345708     chr3 3638059-3638230      + | ENSMUST00000108393       Hnf4g
  ENSMUSE00001345708     chr3 3638059-3638230      + | ENSMUST00000108394       Hnf4g
  ENSMUSE00000149313     chr3 3641223-3641317      + | ENSMUST00000108393       Hnf4g
                 ...      ...             ...    ... .                ...         ...
  ENSMUST00000082414     chrM     10167-11544      + | ENSMUST00000082414      mt-Nd4
  ENSMUST00000082418     chrM     11742-13565      + | ENSMUST00000082418      mt-Nd5
  ENSMUST00000082419     chrM     13552-14070      - | ENSMUST00000082419      mt-Nd6
  ENSMUST00000082421     chrM     14145-15288      + | ENSMUST00000082421     mt-Cytb
  ENSMUST00000084013     chrM      9877-10173      + | ENSMUST00000084013     mt-Nd4l
                                gene_id   gene_biotype     type
                            <character>    <character> <factor>
  ENSMUSE00001236884 ENSMUSG00000017688 protein_coding     exon
  ENSMUSE00000676606 ENSMUSG00000017688 protein_coding     exon
  ENSMUSE00001345708 ENSMUSG00000017688 protein_coding     exon
  ENSMUSE00001345708 ENSMUSG00000017688 protein_coding     exon
  ENSMUSE00000149313 ENSMUSG00000017688 protein_coding     exon
                 ...                ...            ...      ...
  ENSMUST00000082414 ENSMUSG00000064363 protein_coding      cds
  ENSMUST00000082418 ENSMUSG00000064367 protein_coding      cds
  ENSMUST00000082419 ENSMUSG00000064368 protein_coding      cds
  ENSMUST00000082421 ENSMUSG00000064370 protein_coding      cds
  ENSMUST00000084013 ENSMUSG00000065947 protein_coding      cds
  -------
  seqinfo: 22 sequences (1 circular) from mm10 genome
Annotation(data) <- annotations

Computing QC Metrics

# 1. Look at the fragment length periodicity for all the cells
data <- NucleosomeSignal(object = data)
Found 3694 cell barcodes

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# Note:
# Signac looks at all fragments in each cell and bins them by length:
# Fragment length  Category
# 0–100 bp  Nucleosome-free (NFR)
# 180–247 bp    Mononucleosomal
# 315–473 bp    Dinucleosomal
# .........   .............

# Then calculate mononucleosomal/ nucleosome-free ratio

  
data$nucleosome_group <- ifelse(data$nucleosome_signal > 4, 'NS > 4', 'NS < 4')
unique(data$nucleosome_group)
[1] "NS < 4"
# no low-quality cells dominated by mononucleosomal fragments.
FragmentHistogram(object = data, 
                  group.by = 'nucleosome_group', 
                  region = 'chr1-1-10000000')


#Note:
# cells exhibit a pattern that is typical for a successful ATAC-seq experiment.
# 2. Calculate the TSS enrichment score for each cell
data <- TSSEnrichment(data, fast = FALSE)
# 3. Compute fraction of reads from blacklisted region
# in data[[]] there is a column blacklist_region_fragments
# number of fragments mapped to black list regions
data$blacklist_ratio <- data$blacklist_region_fragments / data$peak_region_fragments
data$pct_reads_in_peaks <- data$peak_region_fragments / data$passed_filters * 100 
vln <- VlnPlot(
  object = data,
  features = c('peak_region_fragments', 'pct_reads_in_peaks', 
                'blacklist_ratio', 'nucleosome_signal', 'TSS.enrichment'),
  pt.size = 0.1,
  ncol = 5
)
vln

Remove cells that are outliers for these QC metrics

data
An object of class Seurat 
148897 features across 3694 samples within 1 assay 
Active assay: peaks (148897 features, 0 variable features)
 2 layers present: counts, data
# option 1: Set manual thresholdsbased on the violin plot 
data_filtered <- subset(
  x = data,
  subset = peak_region_fragments > 3000 &
    peak_region_fragments < 40000 &
    pct_reads_in_peaks > 60 &
    blacklist_ratio < 0.02 &
    nucleosome_signal < 4 & 
    TSS.enrichment > 2
)

data_filtered
An object of class Seurat 
148897 features across 3493 samples within 1 assay 
Active assay: peaks (148897 features, 0 variable features)
 2 layers present: counts, data
# Option 2: Based on quantiles
low_prf <- quantile(data[["peak_region_fragments"]]$peak_region_fragments, probs = 0.02)
hig_prf <- quantile(data[["peak_region_fragments"]]$peak_region_fragments, probs = 0.98)

low_prp <- quantile(data[["pct_reads_in_peaks"]]$pct_reads_in_peaks, probs = 0.02)

high_blr <- quantile(data[["blacklist_ratio"]]$blacklist_ratio, probs = 0.98)

hig_ns <- quantile(data[["nucleosome_signal"]]$nucleosome_signal, probs = 0.98)

low_ts <- quantile(data[["TSS.enrichment"]]$TSS.enrichment, probs = 0.02)
print(low_prf)
     2% 
2280.18 
print(hig_prf)
     98% 
22501.22 
print(low_prp)
      2% 
69.90098 
print(high_blr)
       98% 
0.01488525 
print(hig_ns)
      98% 
0.8913645 
print(low_ts)
     2% 
3.73821 
data <- subset(
  x = data,
  subset = peak_region_fragments > low_prf &
    peak_region_fragments < hig_prf &
    pct_reads_in_peaks > low_prp &
    blacklist_ratio < high_blr &
    nucleosome_signal < hig_ns &
    TSS.enrichment > low_ts
)
data
An object of class Seurat 
148897 features across 3300 samples within 1 assay 
Active assay: peaks (148897 features, 0 variable features)
 2 layers present: counts, data

Normalization and linear dimensional reduction

data <- RunTFIDF(data)
data <- FindTopFeatures(data, min.cutoff = 'q0')
data
An object of class Seurat 
148897 features across 3300 samples within 1 assay 
Active assay: peaks (148897 features, 148897 variable features)
 2 layers present: counts, data
data <- RunSVD(data)
DepthCor(data)

# Note:
# Component 1 is strongly correlated to sequencing depth  (technical variation).
# rather than biological variation
# Remove this component

Non-linear dimension reduction and clustering

data <- RunUMAP(
  object = data,
  reduction = 'lsi',
  dims = 2:30
)

data <- FindNeighbors(
  object = data,
  reduction = 'lsi',
  dims = 2:30
)

data <- FindClusters(
  object = data,
  algorithm = 3,
  verbose = FALSE
)
DimPlot(object = data, label = TRUE, pt.size = 1) + NoLegend()

# Using data_filtered

data_filtered <- RunTFIDF(data_filtered)
data_filtered <- FindTopFeatures(data_filtered, min.cutoff = 'q0')
data_filtered <- RunSVD(object = data_filtered)

DepthCor(data_filtered)

data_filtered <- RunUMAP(
  object = data_filtered,
  reduction = 'lsi',
  dims = 2:30
)
Using method 'umap'
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
data_filtered <- FindNeighbors(
  object = data_filtered,
  reduction = 'lsi',
  dims = 2:30
)
data_filtered <- FindClusters(
  object = data_filtered,
  algorithm = 3,
  resolution = 1.2,
  verbose = FALSE
)

DimPlot(object = data_filtered, label = TRUE, pt.size = 1) + NoLegend()

# Note:
# One primary cluster - because data is from aged mice
# Many subclsuters within main cluster

Multiple samples

# Function with all the preprocessing steps and informative print messages
prepare_data <- function(count_path, meta_path, fragment_path){
  
  message("Step 1: Reading count data...")
  counts <- Read10X_h5(count_path)
  message("Count data loaded successfully.")
  
  message("Step 2: Reading metadata...")
  meta <- read.csv(meta_path,
                   header = TRUE,
                   row.names = 1)
  message("Metadata loaded successfully.")
  
  message("Step 3: Creating ChromatinAssay object...")
  chrom_assay <- CreateChromatinAssay(
    counts = counts,
    sep = c(":", "-"),
    genome = 'mm10',
    fragments = fragment_path,
    min.cells = 10,
    min.features = 200
  )
  message("ChromatinAssay created successfully.")
  
  message("Step 4: Creating Seurat object...")
  data <- CreateSeuratObject(
    counts = chrom_assay,
    assay = "peaks",
    meta.data = meta
  )
  message("Seurat object created successfully.")
  
  message("Step 5: Annotating data...")
  Annotation(data) <- annotations
  message("Annotation completed.")
  
  message("Step 6: Computing QC metrics (nucleosome signal, TSS enrichment, blacklist ratio, % reads in peaks)...")
  data <- NucleosomeSignal(object = data)
  data <- TSSEnrichment(object = data, fast = FALSE)
  data$blacklist_ratio <- data$blacklist_region_fragments / data$peak_region_fragments
  data$pct_reads_in_peaks <- data$peak_region_fragments / data$passed_filters * 100
  message("QC metrics computation completed.")
  
  message("Step 7: Calculating filtering thresholds...")
  low_prf <- quantile(data[["peak_region_fragments"]]$peak_region_fragments, probs = 0.02)
  hig_prf <- quantile(data[["peak_region_fragments"]]$peak_region_fragments, probs = 0.98)
  low_prp <- quantile(data[["pct_reads_in_peaks"]]$pct_reads_in_peaks, probs = 0.02)
  high_blr <- quantile(data[["blacklist_ratio"]]$blacklist_ratio, probs = 0.98)
  hig_ns <- quantile(data[["nucleosome_signal"]]$nucleosome_signal, probs = 0.98)
  low_ts <- quantile(data[["TSS.enrichment"]]$TSS.enrichment, probs = 0.02)
  message("Filtering thresholds calculated.")
  
  message("Step 8: Filtering data based on QC thresholds...")
  data <- subset(
    x = data,
    subset = peak_region_fragments > low_prf &
      peak_region_fragments < hig_prf &
      pct_reads_in_peaks > low_prp &
      blacklist_ratio < high_blr &
      nucleosome_signal < hig_ns &
      TSS.enrichment > low_ts
  )
  message("Data filtering completed.")
  
  message("All preprocessing steps completed successfully.")
  
  return(data)
}
aged <- prepare_data("GSE190424_RAW\\Aged\\GSM5723632_Aged_HSC_filtered_peak_bc_matrix.h5",
                     "GSE190424_RAW\\Aged\\GSM5723632_Aged_HSC_singlecell.csv.gz",
                     "GSE190424_RAW\\Aged\\GSM5723632_Aged_HSC_fragments.tsv.gz"
                     )
Checking for 3694 cell barcodes
Found 3694 cell barcodes

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young <- prepare_data("GSE190424_RAW\\Young\\GSM5723631_Young_HSC_filtered_peak_bc_matrix.h5",
                     "GSE190424_RAW\\Young\\GSM5723631_Young_HSC_singlecell.csv.gz",
                     "GSE190424_RAW\\Young\\GSM5723631_Young_HSC_fragments.tsv.gz"
                     )
Checking for 5028 cell barcodes
Found 5028 cell barcodes

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# Add new column to differentiate between sample aged and young
young$sample <- "young"
aged$sample <- "aged"
aged[[]]
young[[]]
combined_data <- merge(young, aged)

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combined_data <- RunTFIDF(combined_data)
combined_data <- FindTopFeatures(combined_data, min.cutoff = 'q0')
combined_data <- RunSVD(object = combined_data)
DepthCor(combined_data)

combined_data <- RunUMAP(object = combined_data, reduction = 'lsi', dims = 2:30)
Using method 'umap'
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
combined_data <- FindNeighbors(object = combined_data, reduction = 'lsi', dims = 2:30)
combined_data <- FindClusters(object = combined_data, verbose = FALSE, algorithm = 3, resolution = .4)
DimPlot(object = combined_data, label = TRUE) + NoLegend()

DimPlot(object = combined_data, label = TRUE, group.by = "sample") + NoLegend()

Data Analysis

# 1. Create a gene activity matrix
# Gene activity based on chromatin accessibility

gene.activities <- GeneActivity(combined_data)

Processed 24766 groups out of 26139. 95% done. Time elapsed: 3s. ETA: 0s.
Processed 26139 groups out of 26139. 100% done. Time elapsed: 3s. ETA: 0s.

  |                                                  | 0 % ~calculating  
  |+++++                                             | 9 % ~03m 26s      
  |++++++++++                                        | 18% ~02m 45s      
  |++++++++++++++                                    | 27% ~02m 31s      
  |+++++++++++++++++++                               | 36% ~02m 20s      
  |+++++++++++++++++++++++                           | 45% ~01m 51s      
  |++++++++++++++++++++++++++++                      | 55% ~01m 26s      
  |++++++++++++++++++++++++++++++++                  | 64% ~01m 14s      
  |+++++++++++++++++++++++++++++++++++++             | 73% ~56s          
  |+++++++++++++++++++++++++++++++++++++++++         | 82% ~36s          
  |++++++++++++++++++++++++++++++++++++++++++++++    | 91% ~18s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=03m 19s

  |                                                  | 0 % ~calculating  
  |+++++                                             | 9 % ~02m 41s      
  |++++++++++                                        | 18% ~01m 57s      
  |++++++++++++++                                    | 27% ~01m 43s      
  |+++++++++++++++++++                               | 36% ~01m 19s      
  |+++++++++++++++++++++++                           | 45% ~01m 16s      
  |++++++++++++++++++++++++++++                      | 55% ~01m 09s      
  |++++++++++++++++++++++++++++++++                  | 64% ~59s          
  |+++++++++++++++++++++++++++++++++++++             | 73% ~45s          
  |+++++++++++++++++++++++++++++++++++++++++         | 82% ~31s          
  |++++++++++++++++++++++++++++++++++++++++++++++    | 91% ~16s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=03m 01s
# gene.activities
# add the gene activity matrix to the Seurat object as a new assay
combined_data[['RNA']] <- CreateAssayObject(counts = gene.activities)
# Normalize
combined_data <- NormalizeData(
  object = combined_data,
  assay = 'RNA',
  normalization.method = 'LogNormalize',
  scale.factor = median(combined_data$nCount_RNA)
)
Performing log-normalization
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
combined_data[["RNA"]]
Assay data with 21821 features for 7792 cells
First 10 features:
 Hnf4g, Zfhx4, Pex2, UBC, 1700008P02Rik, Pkia, Zc2hc1a, Il7, 1700010I02Rik,
Stmn2 
combined_data[["peaks"]]
ChromatinAssay data with 177359 features for 7792 cells
Variable features: 177359 
Genome: mm10 
Annotation present: TRUE 
Motifs present: FALSE 
Fragment files: 2 
combined_data
An object of class Seurat 
199180 features across 7792 samples within 2 assays 
Active assay: peaks (177359 features, 177359 variable features)
 2 layers present: counts, data
 1 other assay present: RNA
 2 dimensional reductions calculated: lsi, umap
DefaultAssay(combined_data)
[1] "peaks"
# Set RNA as default assay 
DefaultAssay(combined_data) <- 'RNA'
# Plit expression of genes in each cluster

FeaturePlot(
  object = combined_data,
  features = c("Hnf4g", "Zfhx4", "Pex2", "Pkia"),
  max.cutoff = 'q95',
)

# Set default assay back to peaks
DefaultAssay(combined_data) <- 'peaks'

Find differentially accessible peaks between clusters

# peak_region_fragments: total number of reads overlapping any peak per cell
# reflects sequencing depth
# latent.vars = 'peak_region_fragments' adjusts for this
# to remove bias from cells with higher/lower depth

da_peaks <- FindMarkers(
  object = combined_data,
  ident.1 = rownames(combined_data[[]][combined_data$sample == "aged",]),
  ident.2 = rownames(combined_data[[]][combined_data$sample == "young",]),
  min.pct = 0.05,
  test.use = 'LR',
  latent.vars = 'peak_region_fragments'
)

  |                                                  | 0 % ~calculating  
  |+                                                 | 1 % ~01h 25m 04s  
  |+                                                 | 2 % ~01h 06m 54s  
  |++                                                | 3 % ~01h 03m 04s  
  |++                                                | 4 % ~59m 17s      
  |+++                                               | 5 % ~58m 02s      
  |+++                                               | 6 % ~56m 07s      
  |++++                                              | 7 % ~55m 25s      
  |++++                                              | 8 % ~54m 12s      
  |+++++                                             | 9 % ~53m 36s      
  |+++++                                             | 10% ~58m 05s      
  |++++++                                            | 11% ~01h 08m 07s  
  |++++++                                            | 12% ~01h 14m 39s  
  |+++++++                                           | 13% ~01h 20m 33s  
  |+++++++                                           | 14% ~01h 33m 41s  
  |++++++++                                          | 15% ~01h 48m 55s  
  |++++++++                                          | 16% ~02h 03m 07s  
  |+++++++++                                         | 17% ~02h 03m 45s  
  |+++++++++                                         | 18% ~02h 12m 26s  
  |++++++++++                                        | 19% ~02h 19m 14s  
  |++++++++++                                        | 20% ~02h 14m 50s  
  |+++++++++++                                       | 21% ~02h 10m 47s  
  |+++++++++++                                       | 22% ~02h 07m 55s  
  |++++++++++++                                      | 23% ~02h 11m 36s  
  |++++++++++++                                      | 24% ~02h 14m 45s  
  |+++++++++++++                                     | 25% ~02h 17m 10s  
  |+++++++++++++                                     | 26% ~02h 18m 22s  
  |++++++++++++++                                    | 27% ~02h 20m 02s  
  |++++++++++++++                                    | 28% ~02h 21m 39s  
  |+++++++++++++++                                   | 29% ~02h 20m 50s  
  |+++++++++++++++                                   | 30% ~02h 18m 44s  
  |++++++++++++++++                                  | 31% ~02h 13m 56s  
  |++++++++++++++++                                  | 32% ~02h 09m 07s  
  |+++++++++++++++++                                 | 33% ~02h 04m 42s  
  |+++++++++++++++++                                 | 34% ~02h 03m 43s  
  |++++++++++++++++++                                | 35% ~02h 02m 43s  
  |++++++++++++++++++                                | 36% ~02h 00m 05s  
  |+++++++++++++++++++                               | 37% ~01h 56m 01s  
  |+++++++++++++++++++                               | 38% ~01h 52m 08s  
  |++++++++++++++++++++                              | 39% ~01h 48m 24s  
  |++++++++++++++++++++                              | 40% ~01h 44m 50s  
  |+++++++++++++++++++++                             | 41% ~01h 41m 25s  
  |+++++++++++++++++++++                             | 42% ~01h 38m 17s  
  |++++++++++++++++++++++                            | 43% ~01h 35m 09s  
  |++++++++++++++++++++++                            | 44% ~01h 32m 10s  
  |+++++++++++++++++++++++                           | 45% ~01h 29m 12s  
  |+++++++++++++++++++++++                           | 46% ~01h 26m 20s  
  |++++++++++++++++++++++++                          | 47% ~01h 23m 35s  
  |++++++++++++++++++++++++                          | 48% ~01h 21m 01s  
  |+++++++++++++++++++++++++                         | 49% ~01h 18m 26s  
  |+++++++++++++++++++++++++                         | 50% ~01h 15m 56s  
  |++++++++++++++++++++++++++                        | 51% ~01h 13m 31s  
  |++++++++++++++++++++++++++                        | 52% ~01h 11m 10s  
  |+++++++++++++++++++++++++++                       | 53% ~01h 08m 53s  
  |+++++++++++++++++++++++++++                       | 54% ~01h 06m 45s  
  |++++++++++++++++++++++++++++                      | 55% ~01h 04m 36s  
  |++++++++++++++++++++++++++++                      | 56% ~01h 02m 30s  
  |+++++++++++++++++++++++++++++                     | 57% ~01h 00m 26s  
  |+++++++++++++++++++++++++++++                     | 58% ~58m 26s      
  |++++++++++++++++++++++++++++++                    | 59% ~56m 29s      
  |++++++++++++++++++++++++++++++                    | 60% ~54m 34s      
  |+++++++++++++++++++++++++++++++                   | 61% ~52m 46s      
  |+++++++++++++++++++++++++++++++                   | 62% ~50m 57s      
  |++++++++++++++++++++++++++++++++                  | 63% ~49m 11s      
  |++++++++++++++++++++++++++++++++                  | 64% ~47m 25s      
  |+++++++++++++++++++++++++++++++++                 | 65% ~45m 41s      
  |+++++++++++++++++++++++++++++++++                 | 66% ~43m 60s      
  |++++++++++++++++++++++++++++++++++                | 67% ~42m 24s      
  |++++++++++++++++++++++++++++++++++                | 68% ~40m 47s      
  |+++++++++++++++++++++++++++++++++++               | 69% ~39m 12s      
  |+++++++++++++++++++++++++++++++++++               | 70% ~37m 39s      
  |++++++++++++++++++++++++++++++++++++              | 71% ~36m 07s      
  |++++++++++++++++++++++++++++++++++++              | 72% ~34m 37s      
  |+++++++++++++++++++++++++++++++++++++             | 73% ~33m 09s      
  |+++++++++++++++++++++++++++++++++++++             | 74% ~31m 45s      
  |++++++++++++++++++++++++++++++++++++++            | 75% ~30m 19s      
  |++++++++++++++++++++++++++++++++++++++            | 76% ~28m 54s      
  |+++++++++++++++++++++++++++++++++++++++           | 77% ~27m 31s      
  |+++++++++++++++++++++++++++++++++++++++           | 78% ~26m 09s      
  |++++++++++++++++++++++++++++++++++++++++          | 79% ~24m 48s      
  |++++++++++++++++++++++++++++++++++++++++          | 80% ~23m 30s      
  |+++++++++++++++++++++++++++++++++++++++++         | 81% ~22m 11s      
  |+++++++++++++++++++++++++++++++++++++++++         | 82% ~20m 53s      
  |++++++++++++++++++++++++++++++++++++++++++        | 83% ~19m 37s      
  |++++++++++++++++++++++++++++++++++++++++++        | 84% ~18m 21s      
  |+++++++++++++++++++++++++++++++++++++++++++       | 85% ~17m 06s      
  |+++++++++++++++++++++++++++++++++++++++++++       | 86% ~15m 52s      
  |++++++++++++++++++++++++++++++++++++++++++++      | 87% ~14m 40s      
  |++++++++++++++++++++++++++++++++++++++++++++      | 88% ~13m 28s      
  |+++++++++++++++++++++++++++++++++++++++++++++     | 89% ~12m 17s      
  |+++++++++++++++++++++++++++++++++++++++++++++     | 90% ~11m 06s      
  |++++++++++++++++++++++++++++++++++++++++++++++    | 91% ~09m 56s      
  |++++++++++++++++++++++++++++++++++++++++++++++    | 92% ~08m 47s      
  |+++++++++++++++++++++++++++++++++++++++++++++++   | 93% ~07m 39s      
  |+++++++++++++++++++++++++++++++++++++++++++++++   | 94% ~06m 32s      
  |++++++++++++++++++++++++++++++++++++++++++++++++  | 95% ~05m 25s      
  |++++++++++++++++++++++++++++++++++++++++++++++++  | 96% ~04m 19s      
  |+++++++++++++++++++++++++++++++++++++++++++++++++ | 97% ~03m 13s      
  |+++++++++++++++++++++++++++++++++++++++++++++++++ | 98% ~02m 08s      
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 99% ~01m 04s      
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=01h 45m 48s
head(da_peaks) # regions that are significant between young and aged samples
plot1 <- VlnPlot(
  object = combined_data,
  features = rownames(da_peaks)[1],             # the peak to plot
  pt.size = 0.1,                   # size of dots per cell
  group.by = "sample"              # groups to split cells by
)

plot2 <- FeaturePlot(
  object = combined_data,
  features = rownames(da_peaks)[1],
  pt.size = 0.1,
  max.cutoff = 'q95'
)
plot1 | plot2

# Find genes nearest to these significant regions
ClosestFeature(combined_data, regions = rownames(da_peaks))
NA
da_peaks$closest_gene <-ClosestFeature(combined_data, regions = rownames(da_peaks))$gene_name
da_peaks$distance <- ClosestFeature(combined_data, regions = rownames(da_peaks))$distance
head(da_peaks)
CoveragePlot(
  object = combined_data,
  region = rownames(da_peaks)[1],
  extend.upstream = 10000,
  extend.downstream = 5000,
  group.by = "sample"
)

CoveragePlot(
  object = combined_data,
  region = rownames(da_peaks)[2],
  extend.upstream = 10000,
  extend.downstream = 5000,
  group.by = "sample"
)

plot1 <- VlnPlot(
  object = combined_data,
  features = rownames(da_peaks)[1],            
  pt.size = 0.1,                   
  group.by = "sample"              
)

plot2 <- FeaturePlot(
  object = combined_data,
  features = rownames(da_peaks)[1],
  pt.size = 0.1,
  max.cutoff = 'q95'
)
plot1 | plot2

plot1 <- VlnPlot(
  object = combined_data,
  features = rownames(da_peaks)[2],            
  pt.size = 0.1,                   
  group.by = "sample"              
)

plot2 <- FeaturePlot(
  object = combined_data,
  features = rownames(da_peaks)[2],
  pt.size = 0.1,
  max.cutoff = 'q95'
)
plot1 | plot2

sessionInfo()
R version 4.4.3 (2025-02-28 ucrt)
Platform: x86_64-w64-mingw32/x64
Running under: Windows 11 x64 (build 26100)

Matrix products: default


locale:
[1] LC_COLLATE=English_India.utf8  LC_CTYPE=English_India.utf8   
[3] LC_MONETARY=English_India.utf8 LC_NUMERIC=C                  
[5] LC_TIME=English_India.utf8    

time zone: Europe/Berlin
tzcode source: internal

attached base packages:
[1] stats4    stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
 [1] future_1.67.0              EnsDb.Mmusculus.v79_2.99.0 ensembldb_2.30.0          
 [4] AnnotationFilter_1.30.0    GenomicFeatures_1.58.0     AnnotationDbi_1.68.0      
 [7] Biobase_2.66.0             GenomicRanges_1.58.0       GenomeInfoDb_1.42.3       
[10] IRanges_2.40.1             S4Vectors_0.44.0           BiocGenerics_0.52.0       
[13] BiocManager_1.30.26        patchwork_1.3.2            ggplot2_4.0.0             
[16] Signac_1.16.0              Seurat_5.3.0               SeuratObject_5.2.0        
[19] sp_2.2-0                   hdf5r_1.3.12              

loaded via a namespace (and not attached):
  [1] RcppAnnoy_0.0.22            splines_4.4.3               later_1.4.4                
  [4] BiocIO_1.16.0               bitops_1.0-9                tibble_3.3.0               
  [7] polyclip_1.10-7             rpart_4.1.24                XML_3.99-0.19              
 [10] fastDummies_1.7.5           lifecycle_1.0.4             globals_0.18.0             
 [13] lattice_0.22-6              MASS_7.3-64                 backports_1.5.0            
 [16] magrittr_2.0.4              sass_0.4.10                 Hmisc_5.2-4                
 [19] plotly_4.11.0               rmarkdown_2.30              jquerylib_0.1.4            
 [22] yaml_2.3.10                 httpuv_1.6.16               sctransform_0.4.2          
 [25] spam_2.11-1                 spatstat.sparse_3.1-0       reticulate_1.43.0          
 [28] cowplot_1.2.0               pbapply_1.7-4               DBI_1.2.3                  
 [31] RColorBrewer_1.1-3          abind_1.4-8                 zlibbioc_1.52.0            
 [34] Rtsne_0.17                  purrr_1.1.0                 biovizBase_1.54.0          
 [37] RCurl_1.98-1.17             nnet_7.3-20                 VariantAnnotation_1.52.0   
 [40] GenomeInfoDbData_1.2.13     ggrepel_0.9.6               irlba_2.3.5.1              
 [43] listenv_0.10.0              spatstat.utils_3.2-0        goftest_1.2-3              
 [46] RSpectra_0.16-2             spatstat.random_3.4-2       fitdistrplus_1.2-4         
 [49] parallelly_1.45.1           codetools_0.2-20            DelayedArray_0.32.0        
 [52] RcppRoll_0.3.1              tidyselect_1.2.1            UCSC.utils_1.2.0           
 [55] farver_2.1.2                base64enc_0.1-3             matrixStats_1.5.0          
 [58] spatstat.explore_3.5-3      GenomicAlignments_1.42.0    jsonlite_2.0.0             
 [61] Formula_1.2-5               progressr_0.17.0            ggridges_0.5.7             
 [64] survival_3.8-3              tools_4.4.3                 ica_1.0-3                  
 [67] Rcpp_1.1.0                  glue_1.8.0                  gridExtra_2.3              
 [70] SparseArray_1.6.2           xfun_0.53                   MatrixGenerics_1.18.1      
 [73] dplyr_1.1.4                 withr_3.0.2                 fastmap_1.2.0              
 [76] digest_0.6.37               R6_2.6.1                    mime_0.13                  
 [79] colorspace_2.1-2            scattermore_1.2             tensor_1.5.1               
 [82] dichromat_2.0-0.1           spatstat.data_3.1-9         RSQLite_2.4.3              
 [85] tidyr_1.3.1                 generics_0.1.4              data.table_1.17.8          
 [88] rtracklayer_1.66.0          httr_1.4.7                  htmlwidgets_1.6.4          
 [91] S4Arrays_1.6.0              uwot_0.2.3                  pkgconfig_2.0.3            
 [94] gtable_0.3.6                blob_1.2.4                  lmtest_0.9-40              
 [97] S7_0.2.0                    XVector_0.46.0              htmltools_0.5.8.1          
[100] dotCall64_1.2               ProtGenerics_1.38.0         scales_1.4.0               
[103] png_0.1-8                   spatstat.univar_3.1-4       knitr_1.50                 
[106] rstudioapi_0.17.1           reshape2_1.4.4              rjson_0.2.23               
[109] checkmate_2.3.3             nlme_3.1-167                curl_7.0.0                 
[112] zoo_1.8-14                  cachem_1.1.0                stringr_1.5.2              
[115] KernSmooth_2.23-26          parallel_4.4.3              miniUI_0.1.2               
[118] foreign_0.8-88              restfulr_0.0.16             pillar_1.11.1              
[121] grid_4.4.3                  vctrs_0.6.5                 RANN_2.6.2                 
[124] promises_1.3.3              xtable_1.8-4                cluster_2.1.8              
[127] htmlTable_2.4.3             evaluate_1.0.5              cli_3.6.5                  
[130] compiler_4.4.3              Rsamtools_2.22.0            rlang_1.1.6                
[133] crayon_1.5.3                future.apply_1.20.0         labeling_0.4.3             
[136] plyr_1.8.9                  stringi_1.8.7               viridisLite_0.4.2          
[139] deldir_2.0-4                BiocParallel_1.40.0         Biostrings_2.74.1          
[142] lazyeval_0.2.2              spatstat.geom_3.6-0         Matrix_1.7-4               
[145] BSgenome_1.74.0             RcppHNSW_0.6.0              bit64_4.6.0-1              
[148] KEGGREST_1.46.0             shiny_1.11.1                SummarizedExperiment_1.36.0
[151] ROCR_1.0-11                 igraph_2.1.4                memoise_2.0.1              
[154] bslib_0.9.0                 fastmatch_1.1-6             bit_4.6.0                  
---
title: "R Notebook"
output: html_notebook
---
```{r}
install.packages("Signac") #Seurat addon for analyzing chromatin
```

```{r}
library(hdf5r) #need to read h5 files
library(Seurat)
library(Signac)
```

```{r}
library(ggplot2)
library(patchwork)
```

```{r}
setwd("C:\\singlecell_atac")
getwd()
```
# Pre-processing workflow

```{r}
counts <- Read10X_h5("GSE190424_RAW\\Aged\\GSM5723632_Aged_HSC_filtered_peak_bc_matrix.h5")
```

```{r}
# counts is a feature by cell matrix, here features are coordinates of peaks.
counts # 167771 features x 3694 cells
```

```{r}
meta <- read.csv("GSE190424_RAW\\Aged\\GSM5723632_Aged_HSC_singlecell.csv.gz",
                 header=TRUE,
                 row.names = 1) 

meta
```


```{r}

  chrom_assay <- CreateChromatinAssay(
  counts = counts,
  sep = c(":", "-"), # features (chr1:3670444-3672753, look in counts) are sep by : and ,
  genome = 'mm10', 
  fragments = 'GSE190424_RAW\\Aged\\GSM5723632_Aged_HSC_fragments.tsv.gz',
  min.cells = 10, # Keep peaks found in ≥10 cells.
  min.features = 200 # Keep cells with ≥200 peaks
)
```

```{r}
# Notes:
# 
# Make sure fragments and its index are in same folder
# Names of both files should match except for the .tbi
# for example: see below
```

```{r}
list.files("GSE190424_RAW\\Aged")
```


```{r}

data <- CreateSeuratObject(
  counts = chrom_assay,
  assay = "peaks",
  meta.data = meta
)
```


```{r}
data
```
```{r}
data[[]]
```

```{r}

if (!require("BiocManager", quietly = TRUE))
    install.packages("BiocManager")

BiocManager::install("EnsDb.Mmusculus.v79") # EnsDb.Hsapiens.v86 for human
BiocManager::install("GenomeInfoDb") #translation between chromosome names
BiocManager::install("biovizBase")
```

```{r}
library(EnsDb.Mmusculus.v79)
library(GenomeInfoDb)
```

```{r}
# get all annotated mouse genes from Ensembl-based annotation database

annotations <- GetGRangesFromEnsDb(ensdb = EnsDb.Mmusculus.v79)
seqlevelsStyle(annotations) <- 'UCSC'

# Note: features have chr1 prefix which is UCSC-style chromosome names. 
```

```{r}
annotations
```

```{r}
Annotation(data) <- annotations
```

# Computing QC Metrics

```{r}
# 1. Look at the fragment length periodicity for all the cells
data <- NucleosomeSignal(object = data)
```
```{r}
# Note:
# Signac looks at all fragments in each cell and bins them by length:
# Fragment length  Category
# 0–100 bp	Nucleosome-free (NFR)
# 180–247 bp	Mononucleosomal
# 315–473 bp	Dinucleosomal
# .........   .............

# Then calculate mononucleosomal/ nucleosome-free ratio

  
```

```{r}
data$nucleosome_group <- ifelse(data$nucleosome_signal > 4, 'NS > 4', 'NS < 4')
unique(data$nucleosome_group)

# no low-quality cells dominated by mononucleosomal fragments.
```

```{r}
FragmentHistogram(object = data, 
                  group.by = 'nucleosome_group', 
                  region = 'chr1-1-10000000')

#Note:
# cells exhibit a pattern that is typical for a successful ATAC-seq experiment.

```

```{r}
# 2. Calculate the TSS enrichment score for each cell
data <- TSSEnrichment(data, fast = FALSE)
```


```{r}
# 3. Compute fraction of reads from blacklisted region
# in data[[]] there is a column blacklist_region_fragments
# number of fragments mapped to black list regions
data$blacklist_ratio <- data$blacklist_region_fragments / data$peak_region_fragments
```

```{r}
data$pct_reads_in_peaks <- data$peak_region_fragments / data$passed_filters * 100 
```


```{r}
vln <- VlnPlot(
  object = data,
  features = c('peak_region_fragments', 'pct_reads_in_peaks', 
                'blacklist_ratio', 'nucleosome_signal', 'TSS.enrichment'),
  pt.size = 0.1,
  ncol = 5
)
vln
```

# Remove cells that are outliers for these QC metrics

```{r}
data
```

```{r}
# option 1: Set manual thresholdsbased on the violin plot 
data_filtered <- subset(
  x = data,
  subset = peak_region_fragments > 3000 &
    peak_region_fragments < 40000 &
    pct_reads_in_peaks > 60 &
    blacklist_ratio < 0.02 &
    nucleosome_signal < 4 & 
    TSS.enrichment > 2
)

data_filtered
```
```{r}
# Option 2: Based on quantiles
low_prf <- quantile(data[["peak_region_fragments"]]$peak_region_fragments, probs = 0.02)
hig_prf <- quantile(data[["peak_region_fragments"]]$peak_region_fragments, probs = 0.98)

low_prp <- quantile(data[["pct_reads_in_peaks"]]$pct_reads_in_peaks, probs = 0.02)

high_blr <- quantile(data[["blacklist_ratio"]]$blacklist_ratio, probs = 0.98)

hig_ns <- quantile(data[["nucleosome_signal"]]$nucleosome_signal, probs = 0.98)

low_ts <- quantile(data[["TSS.enrichment"]]$TSS.enrichment, probs = 0.02)
```

```{r}
print(low_prf)
print(hig_prf)
print(low_prp)
print(high_blr)
print(hig_ns)
print(low_ts)
```

```{r}
data <- subset(
  x = data,
  subset = peak_region_fragments > low_prf &
    peak_region_fragments < hig_prf &
    pct_reads_in_peaks > low_prp &
    blacklist_ratio < high_blr &
    nucleosome_signal < hig_ns &
    TSS.enrichment > low_ts
)
data
```
# Normalization and linear dimensional reduction

```{r}
data <- RunTFIDF(data)
```

```{r}
data <- FindTopFeatures(data, min.cutoff = 'q0')
data
```

```{r}
data <- RunSVD(data)
```

```{r}
DepthCor(data)
```
```{r}
# Note:
# Component 1 is strongly correlated to sequencing depth  (technical variation).
# rather than biological variation
# Remove this component
```

# Non-linear dimension reduction and clustering

```{r}
data <- RunUMAP(
  object = data,
  reduction = 'lsi',
  dims = 2:30
)

data <- FindNeighbors(
  object = data,
  reduction = 'lsi',
  dims = 2:30
)

data <- FindClusters(
  object = data,
  algorithm = 3,
  verbose = FALSE
)

```


```{r}
DimPlot(object = data, label = TRUE, pt.size = 1) + NoLegend()
```

```{r}
# Using data_filtered

data_filtered <- RunTFIDF(data_filtered)
data_filtered <- FindTopFeatures(data_filtered, min.cutoff = 'q0')
data_filtered <- RunSVD(object = data_filtered)

DepthCor(data_filtered)
```

```{r}
data_filtered <- RunUMAP(
  object = data_filtered,
  reduction = 'lsi',
  dims = 2:30
)
data_filtered <- FindNeighbors(
  object = data_filtered,
  reduction = 'lsi',
  dims = 2:30
)
data_filtered <- FindClusters(
  object = data_filtered,
  algorithm = 3,
  resolution = 1.2,
  verbose = FALSE
)

DimPlot(object = data_filtered, label = TRUE, pt.size = 1) + NoLegend()
```

```{r}
# Note:
# One primary cluster - because data is from aged mice
# Many subclsuters within main cluster
```

# Multiple samples

```{r}
# Function with all the preprocessing steps and informative print messages
prepare_data <- function(count_path, meta_path, fragment_path){
  
  message("Step 1: Reading count data...")
  counts <- Read10X_h5(count_path)
  message("Count data loaded successfully.")
  
  message("Step 2: Reading metadata...")
  meta <- read.csv(meta_path,
                   header = TRUE,
                   row.names = 1)
  message("Metadata loaded successfully.")
  
  message("Step 3: Creating ChromatinAssay object...")
  chrom_assay <- CreateChromatinAssay(
    counts = counts,
    sep = c(":", "-"),
    genome = 'mm10',
    fragments = fragment_path,
    min.cells = 10,
    min.features = 200
  )
  message("ChromatinAssay created successfully.")
  
  message("Step 4: Creating Seurat object...")
  data <- CreateSeuratObject(
    counts = chrom_assay,
    assay = "peaks",
    meta.data = meta
  )
  message("Seurat object created successfully.")
  
  message("Step 5: Annotating data...")
  Annotation(data) <- annotations
  message("Annotation completed.")
  
  message("Step 6: Computing QC metrics (nucleosome signal, TSS enrichment, blacklist ratio, % reads in peaks)...")
  data <- NucleosomeSignal(object = data)
  data <- TSSEnrichment(object = data, fast = FALSE)
  data$blacklist_ratio <- data$blacklist_region_fragments / data$peak_region_fragments
  data$pct_reads_in_peaks <- data$peak_region_fragments / data$passed_filters * 100
  message("QC metrics computation completed.")
  
  message("Step 7: Calculating filtering thresholds...")
  low_prf <- quantile(data[["peak_region_fragments"]]$peak_region_fragments, probs = 0.02)
  hig_prf <- quantile(data[["peak_region_fragments"]]$peak_region_fragments, probs = 0.98)
  low_prp <- quantile(data[["pct_reads_in_peaks"]]$pct_reads_in_peaks, probs = 0.02)
  high_blr <- quantile(data[["blacklist_ratio"]]$blacklist_ratio, probs = 0.98)
  hig_ns <- quantile(data[["nucleosome_signal"]]$nucleosome_signal, probs = 0.98)
  low_ts <- quantile(data[["TSS.enrichment"]]$TSS.enrichment, probs = 0.02)
  message("Filtering thresholds calculated.")
  
  message("Step 8: Filtering data based on QC thresholds...")
  data <- subset(
    x = data,
    subset = peak_region_fragments > low_prf &
      peak_region_fragments < hig_prf &
      pct_reads_in_peaks > low_prp &
      blacklist_ratio < high_blr &
      nucleosome_signal < hig_ns &
      TSS.enrichment > low_ts
  )
  message("Data filtering completed.")
  
  message("All preprocessing steps completed successfully.")
  
  return(data)
}

```


```{r}
aged <- prepare_data("GSE190424_RAW\\Aged\\GSM5723632_Aged_HSC_filtered_peak_bc_matrix.h5",
                     "GSE190424_RAW\\Aged\\GSM5723632_Aged_HSC_singlecell.csv.gz",
                     "GSE190424_RAW\\Aged\\GSM5723632_Aged_HSC_fragments.tsv.gz"
                     )
```
```{r}
young <- prepare_data("GSE190424_RAW\\Young\\GSM5723631_Young_HSC_filtered_peak_bc_matrix.h5",
                     "GSE190424_RAW\\Young\\GSM5723631_Young_HSC_singlecell.csv.gz",
                     "GSE190424_RAW\\Young\\GSM5723631_Young_HSC_fragments.tsv.gz"
                     )
```

```{r}
# Add new column to differentiate between sample aged and young
young$sample <- "young"
aged$sample <- "aged"
```

```{r}
aged[[]]
young[[]]
```


```{r}
combined_data <- merge(young, aged)
```

```{r}
combined_data <- RunTFIDF(combined_data)
combined_data <- FindTopFeatures(combined_data, min.cutoff = 'q0')
combined_data <- RunSVD(object = combined_data)
```

```{r}
DepthCor(combined_data)
```

```{r}
combined_data <- RunUMAP(object = combined_data, reduction = 'lsi', dims = 2:30)
combined_data <- FindNeighbors(object = combined_data, reduction = 'lsi', dims = 2:30)
```

```{r}
combined_data <- FindClusters(object = combined_data, verbose = FALSE, algorithm = 3, resolution = .4)
```


```{r}
DimPlot(object = combined_data, label = TRUE) + NoLegend()
```

```{r}
DimPlot(object = combined_data, label = TRUE, group.by = "sample") + NoLegend()
```

# Data Analysis

```{r}
# 1. Create a gene activity matrix or scores
# Estimate gene expression-like values from open chromatin regions (peaks)
# Each entry epresents the gene activity score for that gene in that cell.

gene.activities <- GeneActivity(combined_data)
```

```{r}
# gene.activities
# add the gene activity matrix to the Seurat object as a new assay
combined_data[['RNA']] <- CreateAssayObject(counts = gene.activities)

```

```{r}
# Normalize
combined_data <- NormalizeData(
  object = combined_data,
  assay = 'RNA',
  normalization.method = 'LogNormalize',
  scale.factor = median(combined_data$nCount_RNA)
)
```
```{r}
combined_data[["RNA"]]
```

```{r}
combined_data[["peaks"]]
```
```{r}
combined_data
```


```{r}
DefaultAssay(combined_data)
```
```{r}
# Set RNA as default assay 
DefaultAssay(combined_data) <- 'RNA'
```


```{r}
# Plit expression of genes in each cluster

FeaturePlot(
  object = combined_data,
  features = c("Hnf4g", "Zfhx4", "Pex2", "Pkia"),
  max.cutoff = 'q95',
)
```

```{r}
# Set default assay back to peaks
DefaultAssay(combined_data) <- 'peaks'
```


# Find differentially accessible peaks between clusters

```{r}
# peak_region_fragments: total number of reads overlapping any peak per cell
# reflects sequencing depth
# latent.vars = 'peak_region_fragments' adjusts for peak_region_fragments
# This remove bias from cells with higher/lower depth

# min.pct = 0.05 - only test peaks present in ≥5% of cells in either group

da_peaks <- FindMarkers(
  object = combined_data,
  ident.1 = rownames(combined_data[[]][combined_data$sample == "aged",]),
  ident.2 = rownames(combined_data[[]][combined_data$sample == "young",]),
  min.pct = 0.05,# 
  test.use = 'LR',
  latent.vars = 'peak_region_fragments'
)
```
```{r}
head(da_peaks) # regions that are significant between young and aged samples
```


```{r}
plot1 <- VlnPlot(
  object = combined_data,
  features = rownames(da_peaks)[1],             
  pt.size = 0.1,                   
  group.by = "sample"             
)

plot2 <- FeaturePlot(
  object = combined_data,
  features = rownames(da_peaks)[1],
  pt.size = 0.1,
  max.cutoff = 'q95'
)
plot1 | plot2

```


```{r}
# Find genes nearest to these significant regions
ClosestFeature(combined_data, regions = rownames(da_peaks))

```

```{r}
da_peaks$closest_gene <-ClosestFeature(combined_data, regions = rownames(da_peaks))$gene_name
da_peaks$distance <- ClosestFeature(combined_data, regions = rownames(da_peaks))$distance
```

```{r}
head(da_peaks)
```

```{r}
CoveragePlot(
  object = combined_data,
  region = rownames(da_peaks)[1],
  extend.upstream = 10000,
  extend.downstream = 5000,
  group.by = "sample"
)
```
```{r}
CoveragePlot(
  object = combined_data,
  region = rownames(da_peaks)[2],
  extend.upstream = 10000,
  extend.downstream = 5000,
  group.by = "sample"
)
```

```{r}
plot1 <- VlnPlot(
  object = combined_data,
  features = rownames(da_peaks)[1],            
  pt.size = 0.1,                   
  group.by = "sample"              
)

plot2 <- FeaturePlot(
  object = combined_data,
  features = rownames(da_peaks)[1],
  pt.size = 0.1,
  max.cutoff = 'q95'
)
plot1 | plot2
```


```{r}
plot1 <- VlnPlot(
  object = combined_data,
  features = rownames(da_peaks)[2],            
  pt.size = 0.1,                   
  group.by = "sample"              
)

plot2 <- FeaturePlot(
  object = combined_data,
  features = rownames(da_peaks)[2],
  pt.size = 0.1,
  max.cutoff = 'q95'
)
plot1 | plot2
```
```{r}
sessionInfo()
```

